We propose in vitro studies of the kinetics of formation of native three-dimensional structure in proteins, to find physiologically feasible general conditions for rapid structure formation, and to obtain information on the mechanism(s) of the self-assembly process. We will determine the identity and distribution of disulfide cross-linked intermediates at various stages in the regeneration of reduced, disorganized proteins. Limited proteolysis followed by column chromatographic separations will make it possible to separate disulfide-containing peptides, which will be identified by amino acid composition. The pattern of distribution of disulfides provides a discriminant between a random search assembly process and a process initiated by nucleation followed by limited search of structures. We will test the hypothesis that continuous-chain fragments of proteins represent independent assembly modules. Protein fragments will be subjected to denaturation, renaturation conditions and tested for the formation of native-like structure following the renaturation conditions. Testing will be carried out in several modes, including immunochemical cross-reactivity and substrate specificity. Employing the three-dimensional structural results from X-ray crystallographic studies, we will attempt to devise new generalizations on protein structure suggested by and consistent with the outcome of the experimental work outlined above. BIBLIOGRAPHIC REFERENCES: Johnson, E. R., Oh, K-J., and Wetlaufer, D. B., Formation of Three Dimensional Structure in Protein Fragments. Reactivation of Reduced Hen Egg Lysozyme Fragment 1-127, J. Biol. Chem. 251, 3154-3157 (1976). Anderson, W. L., and Wetlaufer, D. B., The Folding Pathway of Reduced Lysozyme, J. Biol. Chem. 251, 3147-3153 (1976).